Compositions and methods for treating the vertebral column

ABSTRACT

The present invention relates to compositions and methods useful for treating structures of the vertebral column, including vertebral bodies. In one embodiment, a method for promoting bone formation in a vertebral body comprising providing a composition comprising a PDGF solution and a biocompatible matrix and applying the composition to at least one vertebral body. Promoting bone formation in a vertebral body, according to some embodiments, can increase bone volume, mass, and/or density leading to an increase in mechanical strength of the vertebral body treated with a composition of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/631,731, filed on Dec. 4, 2009, now U.S. Pat. No. 9,161,967, which isa continuation-in-part of International Application No.PCT/US2008/065666, filed on Jun. 3, 2008, which claims priority of U.S.Provisional Patent Application Ser. No. 60/933,202, filed Jun. 4, 2007,and 61/026,835 filed Feb. 7, 2008, all of which are incorporated hereinby reference in their entirety. This application is also acontinuation-in-part of International Application No. PCT/US2007/003582and U.S. patent application Ser. No. 11/704,685, now U.S. Pat. No.7,799,754, both of which were filed on Feb. 9, 2007, and both of whichclaim priority of U.S. Provisional Patent Application Ser. No.60/817,988, filed Jun. 30, 2006, and 60/859,809, filed Nov. 17, 2006,all of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods useful fortreating structures of the vertebral column, including vertebral bodies.

BACKGROUND OF THE INVENTION

Musculoskeletal problems are pervasive throughout the population in allage groups and in both sexes. Half of Americans will need services forfractures at some point in their lifetimes according to a widelypublished article presented at the 2003 annual meeting of the AmericanAcademy of Orthopedic Surgeons (AAOS). More than $10 billion per year isspent in the U.S. on hospital care associated with fracture treatmentaccording to this report.

Vertebral compression fractures (VCFs) are the most common osteoporoticfractures, occurring in about 20% of post-menopausal women (Eastell etal., J Bone Miner Res 1991; 6:207-215). It is estimated that 700,000VCFs occur annually, and only 250,000 of these are diagnosed andtreated. Because these fractures are left untreated, osteoporosis mayremain untreated and progress rapidly. Post-menopausal women have a5-fold increased risk of sustaining another vertebral fracture withinthe coming year and 2-fold increased risk of other fragility fractures,including hip fractures (Klotzbuecher et al, J Bone Miner Res, 2000;15:721-739).

VCFs occur when there is a break in one or both of the vertebral bodyend plates, usually due to trauma, causing failure of the anteriorcolumn and weakening the vertebrae from supporting the body duringactivities of daily living. Vertebral compression fractures caused byosteoporosis can cause debilitating back pain, spinal deformity, andheight loss. Both symptomatic and asymptomatic vertebral fractures areassociated with increased morbidity and mortality. With the number ofaged people at risk for osteoporosis is expected to increasedramatically in the coming decades, accurate identification of VCFs andtreatment intervention is necessary to reduce the enormous potentialimpact of this disease on patients and health care systems.

Traditionally, VCFs caused by osteoporosis have been treated with bedrest, narcotic analgesics, braces, and physical therapy. Bed rest,however, leads to accelerated bone loss and physical deconditioning,further aggravating the patient as well as contributing to the problemof osteoporosis. Moreover, the use of narcotics can worsen the mood andmentation problem that may already be prevalent in the elderly.Additionally, brace wear is not well-tolerated by the elderly. Althoughthe current treatments of osteoporosis such as hormone replacement,bisphosphonates, calcitonin, and parathyroid hormone (PTH) analogs dealwith long-term issues, except for calcitonin, they provide no immediatebenefit in terms of pain control once a fracture occurs (Kapuscinski etal., Master Med. Pol. 1996; 28:83-86).

Recently, minimally invasive treatments for vertebral body compressionfractures, vertebroplasty and kyphoplasty, have been developed toaddress the issues of pain and fracture stabilization. Vertebroplasty isthe filling of a fractured vertebral body with the goals of stabilizingthe bone, preventing further collapse, and eliminating acute fracturepain. Vertebroplasty, however, does not attempt to restore vertebralheight and/or sagittal alignment. In addition, because there is no voidin the bone, vertebral filling is performed under less control with lessviscous cement and, as a consequence, filler leaks are common.

Kyphoplasty is a minimally invasive surgical procedure with the goal ofsafety, improving vertebral height and stabilizing VCF. Guided by x-rayimages, an inflatable bone tamp is inflated in the fractured vertebralbody. This compacts the inner cancellous bone as it pushes the fracturedcortices back toward their normal position. Fixation can then be done byfilling the void with a biomaterial under volume control with a moreviscous cement. Although kyphoplasty is considered a safe and effectivetreatment of vertebral compression fractures, biomechanical studiesdemonstrate that cement augmentation places additional stress onadjacent levels. In fact, this increased stiffness can decrease theultimate load to failure of adjacent vertebrae by 8 to 30% and provokesubsequent fractures (Berlemann et al., J Bone Joint Surgery BR, 2002;84:748-52). Compression fracture of one or more vertebral bodiessubsequent to vertebroplasty or kyphoplasty is referred to herein as a“secondary vertebral compression fracture.”

In a recent clinical study, a higher rate of secondary vertebralcompression fracture was observed after kyphoplasty compared withhistorical data for untreated fractures. Most of these occurred at anadjacent level within 2 months of the index procedure. After thistwo-month period, there were only occasional secondary vertebralcompression fractures which occurred at remote levels. This studyconfirmed biomechanical studies showing that cement augmentation placesadditional stress on adjacent level. (Fribourg et al., Incidence ofsubsequent vertebral fracture after kyphoplasty, Spine, 2004; 20;2270-76).

Given the increased incidence of the use of minimally invasive surgicaltechniques for the treatment of vertebral compression fractures, and thepredisposition of adjacent vertebrae to undergo secondary compressionfracture, an unmet clinical need exists to prophylactically treat andprevent secondary VCFs.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods useful fortreating structures of the vertebral column, including vertebral bodies.In some embodiments of the present invention, compositions are providedfor promoting bone formation in a vertebral body. In other embodiments,compositions and methods are provided for preventing or decreasing thelikelihood of vertebral compression fractures. In another embodiment,methods and compositions are provided for preventing or decreasing thelikelihood of secondary vertebral compression fractures associated withvertebroplasty and/or kyphoplasty. The present compositions and methodscan be useful in treating vertebral bodies of compromised patients, suchas those with osteoporosis, diabetes, or other diseases or conditions.

In one aspect, a composition for promoting bone formation in a vertebralbody comprises a solution comprising platelet derived growth factor(PDGF) and biocompatible matrix, wherein the solution is disposed orincorporated in the biocompatible matrix. In some embodiments, the PDGFis absorbed by the biocompatible matrix. In other embodiments, the PDGFis adsorbed onto one or more surfaces of the biocompatible matrix. In afurther embodiment, the PDGF is absorbed by the biocompatible matrix andadsorbed onto one or more surfaces of the biocompatible matrix.

In some embodiments, PDGF is present in the solution in a concentrationranging from about 0.01 mg/ml to about 10 mg/ml, from about 0.05 mg/mlto about 5 mg/ml, from about 0.1 mg/ml to about 1.0 mg/ml, or from about0.2 mg/ml to about 0.4 mg/ml. The concentration of PDGF within thesolution may be within any of the concentration ranges stated above.

In some embodiments of the present invention, PDGF comprises PDGFhomodimers and heterodimers, including PDGF-AA, PDGF-BB, PDGF-AB,PDGF-CC, PDGF-DD, and mixtures and derivatives thereof. In oneembodiment, PDGF comprises PDGF-BB. In another embodiment PDGF comprisesa recombinant human (rh) PDGF such as recombinant human PDGF-BB(rhPDGF-BB).

In some embodiments of the present invention, PDGF comprises PDGFfragments. In one embodiment rhPDGF-B comprises the following fragments:amino acid sequences 1-31, 1-32, 33-108, 33-109, and/or 1-108 of theentire B chain. The complete amino acid sequence (1-109) of the B chainof PDGF is provided in FIG. 15 of U.S. Pat. No. 5,516,896. It is to beunderstood that the rhPDGF compositions of the present invention maycomprise a combination of intact rhPDGF-B (1-109) and fragments thereof.Other fragments of PDGF may be employed such as those disclosed in U.S.Pat. No. 5,516,896. In some embodiments, rhPDGF-BB comprises at least65% of intact rhPDGF-B (1-109).

A biocompatible matrix, according to some embodiments of the presentinvention, comprises a bone substituting agent (also called ascaffolding material herein) and optionally a biocompatible binder. Bonesubstituting agents, in some embodiments, comprise calcium phosphateincluding amorphous calcium phosphate, monocalcium phosphate monohydrate(MCPM), monocalcium phosphate anhydrous (MCPA), dicalcium phosphatedihydrate (DCPD), dicalcium phosphate anhydrous (DCPA), octacalciumphosphate (OCP), α-tricalcium phosphate, β-TCP, hydroxyapatite (OHAp),poorly crystalline hydroxapatite, tetracalcium phosphate (TTCP),heptacalcium decaphosphate, calcium metaphosphate, calcium pyrophosphatedihydrate, calcium pyrophosphate, carbonated calcium phosphate,hydroxyapatite, or derivatives or mixtures thereof. In some embodiments,bone substituting agents comprise calcium sulfate or demineralized bonesuch as dried cortical or cancellous bone.

In another aspect, the present invention provides a composition forpromoting bone formation in a vertebral body comprising a PDGF solutiondisposed in a biocompatible matrix, wherein the biocompatible matrixcomprises a bone scaffolding material and a biocompatible binder. ThePDGF solution may have a concentration of PDGF as described above. Abone scaffolding material, in some embodiments, comprises calciumphosphate. In an embodiment, calcium phosphate comprises β-TCP. In oneaspect, biocompatible matrices may include calcium phosphate particleswith or without biocompatible binders or bone allograft such asdemineralized freeze dried bone allograft (DFDBA), mineralized freezedried bone allograft (FDBA), or particulate demineralized bone matrix(DBM). In another aspect, biocompatible matrices may include boneallograft such as DFDBA, DBM, or other bone allograft materialsincluding cortical bone shapes, such as blocks, wedges, cylinders, orparticles, or cancellous bone particles of various shapes and sizes.

Moreover, a biocompatible binder, according to some embodiments of thepresent invention, comprises proteins, polysaccharides, nucleic acids,carbohydrates, synthetic polymers, or mixtures thereof. In oneembodiment, a biocompatible binder comprises collagen. In anotherembodiment, a biocompatible binder comprises hyaluronic acid.

In another aspect the present invention provides a composition forpreventing or decreasing the likelihood of vertebral compressionfractures, including secondary vertebral compression fractures. In someembodiments, a composition for preventing or decreasing the likelihoodof vertebral compression fractures comprises a solution comprising PDGFand a biocompatible matrix wherein the solution is disposed in thebiocompatible matrix. In other embodiments, a composition for preventingor decreasing the likelihood of vertebral compression fracturescomprises a PDGF solution disposed in a biocompatible matrix, whereinthe biocompatible matrix comprises a bone scaffolding material and abiocompatible binder. In embodiments of a composition for preventing ordecreasing the likelihood of vertebral compression fractures, a PDGFsolution may have a concentration of PDGF as described above. Moreover,a bone scaffolding material, in some embodiments, comprises calciumphosphate. In an embodiment, calcium phosphate comprises β-tricalciumphosphate. A biocompatible binder, according to some embodiments of thepresent invention, comprises proteins, polysaccharides, nucleic acids,carbohydrates, synthetic polymers, or mixtures thereof. In oneembodiment, a biocompatible binder comprises collagen. In anotherembodiment, a biocompatible binder comprises collagen, such as bovinecollagen.

In some embodiments of the present invention, compositions for promotingbone formation in vertebral bodies and compositions for preventing orreducing the likelihood of vertebral compression fractures furthercomprise at least one contrast agent. Contrast agents, according toembodiments of the present invention, are substances operable to atleast partially provide differentiation of two or more bodily tissueswhen imaged. Contrast agents, according to some embodiments, comprisecationic contrast agents, anionic contrast agents, nonionic contrastagents, or mixtures thereof. In some embodiments, contrast agentscomprise radiopaque contrast agents. Radiopaque contrast agents, in someembodiments, comprise iodo-compounds including(S)—N,N′-bis[2-hydroxy-1-(hydroxymethyl)-ethyl]-2,4,6-triiodo-5-lactamidoisophthalamide(Iopamidol) and derivatives thereof.

In another aspect, the present invention provides a kit comprising abiocompatible matrix in a first package and a solution comprising PDGFin a second package. In some embodiments, the biocompatible matrixcomprises a scaffolding material, a scaffolding material and abiocompatible binder, and/or bone allograft such as DFDBA or particulateDBM. In one embodiment, the scaffolding material comprises a calciumphosphate, such as β-TCP. Moreover, in some embodiments, the solutioncomprises a predetermined concentration of PDGF. The concentration ofthe PDGF can be predetermined according to the surgical procedure beingperformed, such as promoting or accelerating bone growth in a vertebralbody or preventing or decreasing the likelihood of secondary vertebralcompression fractures. Moreover, in some embodiments, the biocompatiblematrix can be present in the kit in a predetermined amount. The amountof biocompatible matrix provided by a kit can be dependent on thesurgical procedure being performed. In some embodiments, the secondpackage containing the PDGF solution comprises a syringe. A syringe canfacilitate disposition of the PDGF solution in the biocompatible matrix.Once the PDGF solution has been disposed in the biocompatible matrix, insome embodiments, the resulting composition can placed in a secondsyringe and/or cannula and delivered to a vertebral body.

The present invention also provides methods of producing compositionsfor promoting bone formation in vertebral bodies and preventing ordecreasing the likelihood of compression fractures of vertebral bodies,including secondary vertebral compression fractures. In one embodiment,a method for producing such compositions comprises providing a solutioncomprising PDGF, providing a biocompatible matrix, and disposing thesolution in the biocompatible matrix. In some embodiments, a method ofproducing compositions for promoting bone formation in a vertebral bodyand preventing or decreasing the likelihood of compression fracture in avertebral body further comprises providing a contrast agent anddisposing the contrast agent in the biocompatible matrix.

In another aspect, the present invention provides methods for promotingor accelerating bone formation in a vertebral body comprising providinga composition comprising a PDGF solution disposed in a biocompatiblematrix and applying an effective amount of the composition to at leastone vertebral body. Applying the composition to at least one vertebralbody, in some embodiments, comprises injecting the composition into theat least one vertebral body.

In another aspect, the present invention provides methods comprisingpreventing or decreasing the likelihood of vertebral compressionfractures, including secondary vertebral compression fractures.Preventing or decreasing the likelihood of vertebral compressionfractures, according to embodiments of the present invention comprisesproviding a composition comprising a PDGF solution disposed in abiocompatible matrix and applying an effective amount of the compositionto at least one vertebral body. In some embodiments, applying thecomposition to at least one vertebral body comprises injecting thecomposition into the at least one vertebral body. In one embodiment, thecomposition is applied to a second vertebral body, in some instances anadjacent vertebral body, subsequent to a vertebroplasty or kyphoplastyof a first vertebral body. In some embodiments, a composition comprisinga PDGF solution disposed in a biocompatible matrix is applied to atleast one high risk vertebral body. “High risk vertebral bodies” (HVB),as used herein, refer to vertebral bodies of vertebrae T5 through T12 aswell as L1 through L4, which are at the greatest risk of undergoingsecondary vertebral compression fracture.

In some embodiments of methods of the present invention, thebiocompatible matrix comprises a bone scaffolding material. In someembodiments, the biocompatible matrix comprises a bone scaffoldingmaterial and a biocompatible binder.

In some embodiments, methods for promoting bone formation in vertebralbodies and preventing or decreasing the likelihood of compressionfractures of vertebral bodies further comprise providing at least onepharmaceutical composition in addition to the composition comprising aPDGF solution disposed in a biocompatible matrix and administering theat least one pharmaceutical composition locally and/or systemically. Theat least one pharmaceutical composition, in some embodiments, comprisesvitamins, calcium supplements, or any osteoclast inhibitor known to oneof skill in the art, including bisphosphonates. In some embodiments, theat least one pharmaceutical composition is administered locally. In suchembodiments, the at least one pharmaceutical composition can beincorporated into the biocompatible matrix or otherwise disposed in andaround a vertebral body. In other embodiments, the at least onepharmaceutical composition is administered systemically to a patient. Inone embodiment, for example, the at least one pharmaceutical compositionis administered orally to a patient. In another embodiment, the at leastone pharmaceutical composition is administered intravenously to apatient.

Accordingly, it is an object of the present invention to provide acomposition comprising PDGF useful in promoting bone formation invertebral bodies.

It is another object of the present invention to provide a compositioncomprising PDGF useful in strengthening vertebral bodies.

It is another object of the present invention to provide a compositioncomprising PDGF useful in strengthening vertebral bodies of patientswith osteoporosis.

It is another object of the present invention to provide a compositioncomprising PDGF useful in preventing or decreasing the likelihood ofvertebral compression fractures, including secondary vertebralcompression fractures.

Another object of the present invention is to provide methods forpromoting bone formation in vertebral bodies using compositionscomprising PDGF.

A further object of the present invention is to provide methods ofpreventing or decreasing the likelihood of vertebral compressionfractures, including secondary vertebral compression fractures, usingcompositions comprising PDGF.

These and other embodiments of the present invention are described ingreater detail in the detailed description which follows. These andother objects, features and advantages of the present invention willbecome apparent after a review of the following detailed description ofthe disclosed embodiments and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a syringe and related apparatus penetrating tissueoverlaying a vertebral body to deliver a composition of the presentinvention to the vertebral body according to an embodiment of thepresent invention.

FIG. 2 is a radiograph illustrating injection of a composition into avertebral body according to an embodiment of the present invention.

FIG. 3 illustrates vertebrae receiving compositions of the presentinvention according to one embodiment of the present invention.

FIG. 4 illustrates percent change in volumetric bone mineral density forvertebral bodies receiving a composition comprising 1.0 mg/ml ofrhPDGF-BB disposed in β-TCP/collagen matrix in comparison with vertebralbodies receiving a composition comprising 20 mM sodium acetate bufferdisposed in β-TCP/collagen matrix according to one embodiment of thepresent invention.

FIG. 5 illustrates percent change in volumetric bone mineral density forvertebral bodies receiving a composition comprising 1.0 mg/ml ofrhPDGF-BB disposed in β-TCP/collagen matrix in comparison with vertebralbodies receiving a composition comprising 20 mM sodium acetate bufferdisposed in β-TCP/collagen matrix according to one embodiment of thepresent invention.

DETAILED DESCRIPTION

The present invention provides compositions and methods useful fortreating structures of the vertebral column, including vertebral bodies.According to embodiments described herein, the present inventionprovides compositions for promoting bone formation in a vertebral bodyand compositions for preventing or decreasing the likelihood ofvertebral compression fractures, including secondary vertebralcompression fractures. In one embodiment, the compositions comprise asolution comprising PDGF and a biocompatible matrix, wherein thesolution is disposed in the biocompatible matrix. In another embodiment,the compositions comprise a PDGF solution disposed in a biocompatiblematrix, wherein the biocompatible matrix comprises a bone scaffoldingmaterial and a biocompatible binder. In one aspect, biocompatiblematrices include calcium phosphate particles with or withoutbiocompatible binders or bone allograft such as DFDBA or particulateDBM. In another aspect, biocompatible matrices may include DFDBA or DBM.

Turning now to components that can be included in various embodiments ofthe present invention, compositions of the present invention comprise asolution comprising PDGF.

PDGF Solutions

PDGF plays an important role in regulating cell growth and migration.PDGF, as with other growth factors, binds with the extracellular domainsof receptor tyrosine kinases. The binding of PDGF to these transmembraneproteins activate the kinase activity of their catalytic domains locatedon the cytosolic side of the membrane. By phosphorylating tyrosineresidues of target proteins, the kinases induce a variety of cellularprocesses that include cell growth and extracellular matrix production.

In one aspect, a composition provided by the present invention comprisesa solution comprising PDGF and a biocompatible matrix, wherein thesolution is disposed or incorporated in the biocompatible matrix. Insome embodiments, PDGF is present in the solution in a concentrationranging from about 0.01 mg/ml to about 10 mg/ml, from about 0.05 mg/mlto about 5 mg/ml, or from about 0.1 mg/ml to about 1.0 mg/ml. PDGF maybe present in the solution at any concentration within these statedranges including the upper limit and lower limit of each range. In otherembodiments, PDGF is present in the solution at any one of the followingconcentrations: about 0.05 mg/ml; about 0.1 mg/ml; about 0.15 mg/ml;about 0.2 mg/ml; about 0.25 mg/ml; about 0.3 mg/ml; about 0.35 mg/ml;about 0.4 mg/ml; about 0.45 mg/ml; about 0.5 mg/ml, about 0.55 mg/ml,about 0.6 mg/ml, about 0.65 mg/ml, about 0.7 mg/ml; about 0.75 mg/ml;about 0.8 mg/ml; about 0.85 mg/ml; about 0.9 mg/ml; about 0.95 mg/ml; orabout 1.0 mg/ml. In some embodiments, PDGF is present in the solution ina concentration ranging from about 0.2 mg/ml to about 2 mg/ml, fromabout 0.3 mg/ml to about 3 mg/ml, from about 0.4 mg/ml to about 4 mg/ml,or from about 0.5 mg/ml to about 5 mg/ml. It is to be understood thatthese concentrations are simply examples of particular embodiments, andthat the concentration of PDGF may be within any of the concentrationranges stated above including the upper limit and the lower limit ofeach range.

Various amounts of PDGF may be used in the compositions of the presentinvention. Amounts of PDGF that could be used include amounts in thefollowing ranges: about 1 μg to about 50 mg, about 10 μg to about 25 mg,about 100 μg to about 10 mg, and about 250 μg to about 5 mg.

The concentration of PDGF or other growth factors in embodiments of thepresent invention can be determined by using an enzyme-linkedimmunoassay as described in U.S. Pat. Nos. 6,221,625, 5,747,273, and5,290,708, or any other assay known in the art for determining PDGFconcentration. When provided herein, the molar concentration of PDGF isdetermined based on the molecular weight (MW) of PDGF dimer (e.g.,PDGF-BB; MW about 25 kDa).

In some embodiments of the present invention, PDGF comprises PDGFhomodimers and heterodimers, including PDGF-AA, PDGF-BB, PDGF-AB,PDGF-CC, PDGF-DD, and mixtures and derivatives thereof. In oneembodiment, for example, PDGF comprises PDGF-BB. In another embodimentPDGF comprises a recombinant human PDGF, such as rhPDGF-BB. In someembodiments, PDGF comprises mixtures of the various homodimers and/orheterodimers. Embodiments of the present invention contemplate anycombination of PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CC, and/or PDGF-DD.

PDGF, in some embodiments, can be obtained from natural sources. Inother embodiments, PDGF can be produced by recombinant DNA techniques.In other embodiments, PDGF or fragments thereof may be produced usingpeptide synthesis techniques known to one of ordinary skill in the art,such as solid phase peptide synthesis. When obtained from naturalsources, PDGF can be derived from biological fluids. Biological fluids,according to some embodiments, can comprise any treated or untreatedfluid associated with living organisms including blood.

Biological fluids, in another embodiment, can also comprise bloodcomponents including platelet concentrate (PC), apheresed platelets,platelet-rich plasma (PRP), plasma, serum, fresh frozen plasma (FFP),and buffy coat (BC). Biological fluids, in a further embodiment, cancomprise platelets separated from plasma and resuspended in aphysiological fluid.

When produced by recombinant DNA techniques, a DNA sequence encoding asingle monomer (e.g., PDGF B-chain or A-chain), in some embodiments, canbe inserted into cultured prokaryotic or eukaryotic cells for expressionto subsequently produce the homodimer (e.g. PDGF-BB or PDGF-AA). Inother embodiments, a PDGF heterodimer can be generated by inserting DNAsequences encoding for both monomeric units of the heterodimer intocultured prokaryotic or eukaryotic cells and allowing the translatedmonomeric units to be processed by the cells to produce the heterodimer(e.g. PDGF-AB). Commercially available GMP recombinant PDGF-BB can beobtained commercially from Novartis Corporation (Emeryville, Calif.).Research grade rhPDGF-BB can be obtained from multiple sources includingR&D Systems, Inc. (Minneapolis, Minn.), BD Biosciences (San Jose,Calif.), and Chemicon, International (Temecula, Calif.). In someembodiments, monomeric units can be produced in prokaryotic cells in adenatured form, wherein the denatured form is subsequently refolded intoan active molecule.

In embodiments of the present invention, PDGF comprises PDGF fragments.In one embodiment rhPDGF-B comprises the following fragments: amino acidsequences 1-31, 1-32, 33-108, 33-109, and/or 1-108 of the entire Bchain. The complete amino acid sequence (1-109) of the B chain of PDGFis provided in FIG. 15 of U.S. Pat. No. 5,516,896. It is to beunderstood that the rhPDGF compositions of the present invention maycomprise a combination of intact rhPDGF-B (1-109) and fragments thereof.Other fragments of PDGF may be employed such as those disclosed in U.S.Pat. No. 5,516,896. In accordance with one embodiment, the rhPDGF-BBcomprises at least 60% of intact rhPDGF-B (1-109). In anotherembodiment, the rhPDGF-BB comprises at least 65%, 75%, 80%, 85%, 90%,95%, or 99% of intact rhPDGF-B (1-109).

In some embodiments of the present invention, PDGF can be purified.Purified PDGF, as used herein, comprises compositions having greaterthan about 95% by weight PDGF prior to incorporation in solutions of thepresent invention. The solution may be any pharmaceutically acceptablesolution. In other embodiments, the PDGF can be substantially purified.Substantially purified PDGF, as used herein, comprises compositionshaving about 5% to about 95% by weight PDGF prior to incorporation intosolutions of the present invention. In one embodiment, substantiallypurified PDGF comprises compositions having about 65% to about 95% byweight PDGF prior to incorporation into solutions of the presentinvention. In other embodiments, substantially purified PDGF comprisescompositions having about 70% to about 95%, about 75% to about 95%,about 80% to about 95%, about 85% to about 95%, or about 90% to about95%, by weight PDGF, prior to incorporation into solutions of thepresent invention. Purified PDGF and substantially purified PDGF may beincorporated into scaffolds and binders.

In a further embodiment, PDGF can be partially purified. Partiallypurified PDGF, as used herein, comprises compositions having PDGF in thecontext of platelet rich plasma (PRP), fresh frozen plasma (FFP), or anyother blood product that requires collection and separation to producePDGF. Embodiments of the present invention contemplate that any of thePDGF isoforms provided herein, including homodimers and heterodimers,can be purified or partially purified. Compositions of the presentinvention containing PDGF mixtures may contain PDGF isoforms or PDGFfragments in partially purified proportions. Partially purified andpurified PDGF, in some embodiments, can be prepared as described in U.S.patent application Ser. No. 11/159,533 (Publication No: 20060084602).

In some embodiments, solutions comprising PDGF are formed bysolubilizing PDGF in one or more buffers. Buffers suitable for use inPDGF solutions of the present invention can comprise, but are notlimited to, carbonates, phosphates (e.g. phosphate buffered saline),histidine, acetates (e.g. sodium acetate), acidic buffers such as aceticacid and HCl, and organic buffers such as lysine, Tris buffers (e.g.tris(hydroxymethyl)aminoethane),N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and3-(N-morpholino) propanesulfonic acid (MOPS). Buffers can be selectedbased on biocompatibility with PDGF and the buffer's ability to impedeundesirable protein modification. Buffers can additionally be selectedbased on compatibility with host tissues. In one embodiment, sodiumacetate buffer is used. The buffers may be employed at differentmolarities, for example, about 0.1 mM to about 100 mM, about 1 mM toabout 50 mM, about 5 mM to about 40 mM, about 10 mM to about 30 mM, orabout 15 mM to about 25 mM, or any molarity within these ranges. In someembodiments, an acetate buffer is employed at a molarity of about 20 mM.

In another embodiment, solutions comprising PDGF are formed bysolubilizing lyophilized PDGF in water, wherein prior to solubilizationthe PDGF is lyophilized from an appropriate buffer.

Solutions comprising PDGF, according to embodiments of the presentinvention, can have a pH ranging from about 3.0 to about 8.0. In oneembodiment, a solution comprising PDGF has a pH ranging from about 5.0to about 8.0, from about 5.5 to about 7.0, or from about 5.5 to about6.5, or any value within these ranges. The pH of solutions comprisingPDGF, in some embodiments, can be compatible with the prolongedstability and efficacy of PDGF or any other desired biologically activeagent. PDGF is more stable in an acidic environment. Therefore, inaccordance with one embodiment the present invention comprises an acidicstorage formulation of a PDGF solution. In accordance with thisembodiment, the PDGF solution preferably has a pH from about 3.0 toabout 7.0 or from about 4.0 to about 6.5. The biological activity ofPDGF, however, can be optimized in a solution having a neutral pH range.Therefore, in a further embodiment, the present invention comprises aneutral pH formulation of a PDGF solution. In accordance with thisembodiment, the PDGF solution preferably has a pH from about 5.0 toabout 8.0, from about 5.5 to about 7.0, or from about 5.5 to about 6.5.In accordance with a method of the present invention, an acidic PDGFsolution is reformulated to a neutral pH composition, wherein suchcomposition is then used to treat bone and promote bone growth and/orhealing. In accordance with some embodiments of the present invention,the PDGF utilized in the solutions is rh-PDGF-BB. In a furtherembodiment, the pH of the PDGF containing solution may be altered tooptimize the binding kinetics of PDGF to a matrix substrate or linker.If desired, the pH of the material equilibrates to adjacent material,the bound PDGF may become labile.

The pH of solutions comprising PDGF, in some embodiments, can becontrolled by the buffers recited herein. Various proteins demonstratedifferent pH ranges in which they are stable. Protein stabilities areprimarily reflected by isoelectric points and charges on the proteins.The pH range can affect the conformational structure of a protein andthe susceptibility of a protein to proteolytic degradation, hydrolysis,oxidation, and other processes that can result in modification to thestructure and/or biological activity of the protein.

In some embodiments, solutions comprising PDGF can further compriseadditional components, such as other biologically active agents. Inother embodiments, solutions comprising PDGF can further comprise cellculture media, other stabilizing proteins such as albumin, antibacterialagents, protease inhibitors [e.g., ethylenediaminetetraacetic acid(EDTA), ethylene glycol-bis(beta-aminoethylether)-N, N,N′,N′-tetraaceticacid (EGTA), aprotinin, ε-aminocaproic acid (EACA), etc.] and/or othergrowth factors such as fibroblast growth factors (FGFs), epidermalgrowth factors (EGFs), transforming growth factors (TGFs), keratinocytegrowth factors (KGFs), insulin-like growth factors (IGFs), hepatocytegrowth factors (HGFs), bone morphogenetic proteins (BMPs), or otherPDGFs including compositions of PDGF-AA, PDGF-BB, PDGF-AB, PDGF-CCand/or PDGF-DD.

In addition to solutions comprising PDGF, compositions of the presentinvention also comprise a biocompatible matrix in which to dispose thePDGF solutions and may also comprise a biocompatible binder either withor without addition of a biocompatible matrix.

Biocompatible Matrix

Bone Scaffolding Material

A biocompatible matrix, according to embodiments of the presentinvention, comprises a bone scaffolding material. It is to be understoodthat the terms bone scaffolding material and bone substituting agent areused interchangeably in the present application. The bone scaffoldingmaterial provides the framework or scaffold for new bone and tissuegrowth to occur. In some embodiments, a bone scaffolding material hasmultidirectional and interconnected pores of varying diameters. In someembodiments, a bone scaffolding material comprises a plurality ofpockets and non-interconnected pores in addition to the interconnectedpores. A bone scaffolding material, in some embodiments, is one that canpermanently or temporarily replace bone. Following implantation, thebone scaffolding material can be retained by the body or it can beresorbed by the body and replaced by bone.

A bone scaffolding material, in some embodiments, comprises at least onecalcium phosphate. In other embodiments, a bone scaffolding material cancomprise a plurality of calcium phosphates. Calcium phosphates suitablefor use as a bone scaffolding material, in embodiments of the presentinvention, have a calcium to phosphorus atomic ratio ranging from 0.5 to2.0. In some embodiments, a bone scaffolding material comprises anallograft such as DFDBA, FDBA, or particulate DBM. In some embodiments,a bone scaffolding material comprises mineralized bone allograft,mineralized bone, mineralized deproteinized xenograft, or demineralizedbone.

Non-limiting examples of calcium phosphates suitable for use as bonescaffolding materials comprise amorphous calcium phosphate, monocalciumphosphate monohydrate (MCPM), monocalcium phosphate anhydrous (MCPA),dicalcium phosphate dihydrate (DCPD), dicalcium phosphate anhydrous(DCPA), octacalcium phosphate (OCP), α-tricalcium phosphate, β-TCP,hydroxyapatite (OHAp), poorly crystalline hydroxapatite, tetracalciumphosphate (TTCP), heptacalcium decaphosphate, calcium metaphosphate,calcium pyrophosphate dihydrate, calcium pyrophosphate, carbonatedcalcium phosphate, hydroxyapatite, or derivatives or mixtures thereof.

In some embodiments, a bone scaffolding material comprises a polymericmaterial. A polymeric scaffold, in some embodiments, comprises collagen,polylactic acid, poly(L-lactide), poly(D,L-lactide), polyglycolic acid,poly(L-lactide-co-glycolide), poly(L-lactide-co-D,L-lactide),polyacrylate, polymethacrylate, polymethylmethacrylate, chitosan, orcombinations or derivatives thereof.

In some embodiments, a bone scaffolding material comprises porousstructure. Porosity is a desirable characteristic as it facilitates cellmigration and infiltration into the scaffolding material so that theinfiltrating cells can secrete extracellular bone matrix. Porosity alsoprovides access for vascularization. Porosity also provides a highsurface area for enhanced resorption and release of active substances aswell as increased cell-matrix interaction. A bone scaffolding material,in some embodiments, can be sized and shaped prior to use. In someembodiments, the bone scaffolding material can be provided in a shapesuitable for implantation.

Porous bone scaffolding materials, according to some embodiments, cancomprise pores having diameters ranging from about 1 μm to about 1 mm.In one embodiment, a bone scaffolding material comprises macroporeshaving diameters ranging from about 100 μm to about 1 mm or greater. Inanother embodiment, a bone scaffolding material comprises mesoporeshaving diameters ranging from about 10 μm to about 100 μm. In a furtherembodiment, a bone scaffolding material comprises micropores havingdiameters less than about 10 μm. Embodiments of the present inventioncontemplate bone scaffolding materials comprising macropores, mesopores,and micropores or any combination thereof.

A porous bone scaffolding material, in one embodiment, has a porositygreater than about 25% or greater than about 40%. In another embodiment,a porous bone scaffolding material has a porosity greater than about50%, greater than about 60%, greater than about 65%, greater than about70%, greater than about 80%, or greater than about 85%. In a furtherembodiment, a porous bone scaffolding material has a porosity greaterthan about 90%. In some embodiments, a porous bone scaffolding materialcomprises a porosity that facilitates cell migration into thescaffolding material.

In some embodiments, a bone scaffolding material comprises a pluralityof particles. A bone scaffolding material, for example, can comprise aplurality of calcium phosphate particles. Particles of a bonescaffolding material, in some embodiments, can individually demonstrateany of the pore diameters and porosities provided here for the bonescaffolding material. In other embodiments, particles of a bonescaffolding material can form an association to produce a matrix havingany of the pore diameters or porosities provided herein for the bonescaffolding material.

Bone scaffolding particles may be mm, μm, or submicron (nm) in size.Bone scaffolding particles, in one embodiment, have an average diameterranging from about 1 μm to about 5 mm. In other embodiments, particleshave an average diameter ranging from about 1 mm to about 2 mm, fromabout 1 mm to about 3 mm, or from about 250 μm to about 750 μm. Bonescaffolding particles, in another embodiment, have an average diameterranging from about 100 μm to about 300 μm. In a further embodiment, bonescaffolding particles have an average diameter ranging from about 75 μmto about 300 μm. In additional embodiments, bone scaffolding particleshave an average diameter less than about 25 μm, less than about 1 μm, orless than about 1 mm. In some embodiments, scaffolding particles have anaverage diameter ranging from about 100 μm to about 5 mm or from about100 μm to about 3 mm. In other embodiments, bone scaffolding particleshave an average diameter ranging from about 250 μm to about 2 mm, fromabout 250 μm to about 1 mm, or from about 200 μm to about 3 mm.Particles may also be in the range of about 1 nm to about 1 μm, lessthan about 500 nm, or less than about 250 nm.

Bone scaffolding materials, according to some embodiments, can beprovided in a shape suitable for implantation (e.g., a sphere, acylinder, or a block). In other embodiments, bone scaffolding materialsare moldable, extrudable and/or injectable. Moldable, extrudable, andinjectable bone scaffolding materials can facilitate efficient placementof compositions of the present invention in and around vertebral bodies.In some embodiments, bone scaffolding materials are flowable. Flowablebone scaffolding materials, in some embodiments, can be appliedvertebral bodies through a syringe and needle or cannula. In someembodiments, bone scaffolding materials harden in vivo.

In some embodiments, bone scaffolding materials are bioresorbable. Abone scaffolding material, in one embodiment, can be at least 30%, 40%,50%, 60%, 70%, 75%, or 90% resorbed within one year subsequent to invivo implantation. In another embodiment, a bone scaffolding materialcan be resorbed at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, or90% within 1, 3, 6, 9, 12, or 18 months of in vivo implantation. In someembodiments, a bone scaffolding material is greater than 90% resorbedwithin 1, 3, 6, 9, 12, or 18 months of in vivo implantation.Bioresorbability will be dependent on: (1) the nature of the matrixmaterial (i.e., its chemical make up, physical structure and size); (2)the location within the body in which the matrix is placed; (3) theamount of matrix material that is used; (4) the metabolic state of thepatient (diabetic/non-diabetic, osteoporotic, smoker, old age, steroiduse, etc.); (5) the extent and/or type of injury treated; and (6) theuse of other materials in addition to the matrix such as other boneanabolic, catabolic and anti-catabolic factors.

Bone Scaffolding Comprising β-Tricalcium Phosphate (β-TCP)

A bone scaffolding material for use as a biocompatible matrix cancomprise β-TCP. β-TCP, according to some embodiments, can comprise aporous structure having multidirectional and interconnected pores ofvarying diameters. In some embodiments, β-TCP comprises a plurality ofpockets and non-interconnected pores of various diameters in addition tothe interconnected pores. The porous structure of β-TCP, in oneembodiment, comprises macropores having diameters ranging from about 100μm to about 1 mm or greater, mesopores having diameters ranging fromabout 10 μm to about 100 μm, and micropores having diameters less thanabout 10 μm. Macropores and micropores of the β-TCP can facilitateosteoinduction and osteoconduction while macropores, mesopores andmicropores can permit fluid communication and nutrient transport tosupport bone regrowth throughout the β-TCP biocompatible matrix.

In comprising a porous structure, β-TCP, in some embodiments, can have aporosity greater than 25% or greater than about 40%. In otherembodiments, β-TCP can have a porosity greater than 50%, greater thanabout 60%, greater than about 65%, greater than about 70%, greater thanabout 75%, greater than about 80%, or greater than about 85%. In afurther embodiment, β-TCP can have a porosity greater than 90%. In someembodiments, β-TCP can have a porosity that facilitates cell migrationinto the β-TCP.

In some embodiments, a β-TCP bone scaffolding material comprises β-TCPparticles. β-TCP particles, in some embodiments, can individuallydemonstrate any of the pore diameters, pore structures, and porositiesprovided herein for scaffolding materials.

β-TCP particles, in one embodiment have an average diameter ranging fromabout 1 μm to about 5 mm. In other embodiments, β-TCP particles have anaverage diameter ranging from about 1 mm to about 2 mm, from about 1 mmto about 3 mm, from about 100 μm to about 5 mm, from about 100 μm toabout 3 mm, from about 250 μm to about 2 mm, from about 250 μm to about750 μm, from about 250 μm to about 1 mm, from about 250 μm to about 2mm, or from about 200 μm to about 3 mm. In another embodiment, β-TCPparticles have an average diameter ranging from about 100 μm to about300 μm. In some embodiments, β-TCP particles have an average diameterranging from about 75 μm to about 300 μm. In some embodiments, β-TCPparticles have an average diameter of less than about 25 μm, less thanabout 1 μm, or less than about 1 mm. In some embodiments, β-TCPparticles have an average diameter ranging from about 1 nm to about 1μm. In a further embodiment, β-TCP particles have an average diameterless than about 500 nm or less than about 250 nm.

A biocompatible matrix comprising a β-TCP bone scaffolding material, insome embodiments, can be provided in a shape suitable for implantation(e.g., a sphere, a cylinder, or a block). In other embodiments, a β-TCPbone scaffolding material can be moldable, extrudable, and/or flowablethereby facilitating application of the matrix to vertebral bodies.Flowable matrices may be applied through syringes, tubes, cannulas, orspatulas.

A β-TCP bone scaffolding material, according to some embodiments, isbioresorbable. In one embodiment, a β-TCP bone scaffolding material canbe at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, or 85% resorbed oneyear subsequent to in vivo implantation. In another embodiment, a β-TCPbone scaffolding material can be greater than 90% resorbed one yearsubsequent to in vivo implantation.

Bone Scaffolding Material and Biocompatible Binder

In another embodiment, a biocompatible matrix comprises a bonescaffolding material and a biocompatible binder. Bone scaffoldingmaterials in embodiments of a biocompatible matrix further comprising abiocompatible binder are consistent with those provided hereinabove.

Biocompatible binders, according to some embodiments, can comprisematerials operable to promote cohesion between combined substances. Abiocompatible binder, for example, can promote adhesion betweenparticles of a bone scaffolding material in the formation of abiocompatible matrix. In certain embodiments, the same material mayserve as both a scaffolding material. In some embodiments, for example,polymeric materials described herein such as collagen and chitosan mayserve as both scaffolding material and a binder.

Biocompatible binders, in some embodiments, can comprise collagen,polysaccharides, nucleic acids, carbohydrates, proteins, polypeptides,poly(α-hydroxy acids), poly(lactones), poly(amino acids),poly(anhydrides), polyurethanes, poly(orthoesters),poly(anhydride-co-imides), poly(orthocarbonates), poly(α-hydroxyalkanoates), poly(dioxanones), poly(phosphoesters), polylactic acid,poly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA), polyglycolide (PGA),poly(lactide-co-glycolide (PLGA), poly(L-lactide-co-D,L-lactide),poly(D,L-lactide-co-trimethylene carbonate), polyglycolic acid,polyhydroxybutyrate (PHB), poly(ε-caprolactone), poly(δ-valerolactone),poly(γ-butyrolactone), poly(caprolactone), polyacrylic acid,polycarboxylic acid, poly(allylamine hydrochloride),poly(diallyldimethylammonium chloride), poly(ethyleneimine),polypropylene fumarate, polyvinyl alcohol, polyvinylpyrrolidone,polyethylene, polymethylmethacrylate, carbon fibers, poly(ethyleneglycol), poly(ethylene oxide), poly(vinyl alcohol),poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethyleneoxide)-co-poly(propylene oxide) block copolymers, poly(ethyleneterephthalate)polyamide, and copolymers and mixtures thereof.

Biocompatible binders, in other embodiments, can comprise alginic acid,arabic gum, guar gum, xantham gum, gelatin, chitin, chitosan, chitosanacetate, chitosan lactate, chondroitin sulfate, N,O-carboxymethylchitosan, a dextran (e.g., α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin, or sodium dextran sulfate), fibrin glue, lecithin,phosphatidylcholine derivatives, glycerol, hyaluronic acid, sodiumhyaluronate, a cellulose (e.g., methylcellulose, carboxymethylcellulose,hydroxypropyl methylcellulose, or hydroxyethyl cellulose), aglucosamine, a proteoglycan, a starch (e.g., hydroxyethyl starch orstarch soluble), lactic acid, pluronic acids, sodium glycerophosphate,glycogen, a keratin, silk, and derivatives and mixtures thereof.

In some embodiments, a biocompatible binder is water-soluble. Awater-soluble binder can dissolve from the biocompatible matrix shortlyafter its implantation, thereby introducing macroporosity into thebiocompatible matrix. Macroporosity, as discussed herein, can increasethe osteoconductivity of the implant material by enhancing the accessand, consequently, the remodeling activity of the osteoclasts andosteoblasts at the implant site.

In some embodiments, a biocompatible binder can be present in abiocompatible matrix in an amount ranging from about 5 weight percent toabout 50 weight percent of the matrix. In other embodiments, abiocompatible binder can be present in an amount ranging from about 10weight percent to about 40 weight percent of the biocompatible matrix.In another embodiment, a biocompatible binder can be present in anamount ranging from about 15 weight percent to about 35 weight percentof the biocompatible matrix. In a further embodiment, a biocompatiblebinder can be present in an amount of about 20 weight percent of thebiocompatible matrix. In another embodiment, a biocompatible binder canbe present in a biocompatible matrix in an amount greater than about 50weight percent or 60 weight percent of the matrix. In one embodiment, abiocompatible binder can be present in a biocompatible matrix in anamount up to about 99 weight percent of the matrix.

A biocompatible matrix comprising a bone scaffolding material and abiocompatible binder, according to some embodiments, can be flowable,moldable, and/or extrudable. In such embodiments, a biocompatible matrixcan be in the form of a paste or putty. A biocompatible matrix in theform of a paste or putty, in one embodiment, can comprise particles of abone scaffolding material adhered to one another by a biocompatiblebinder.

A biocompatible matrix in paste or putty form can be molded into thedesired implant shape or can be molded to the contours of theimplantation site. In one embodiment, a biocompatible matrix in paste orputty form can be injected into an implantation site with a syringe orcannula.

In some embodiments, a biocompatible matrix in paste or putty form doesnot harden and retains a flowable and moldable form subsequent toimplantation. In other embodiments, a paste or putty can hardensubsequent to implantation, thereby reducing matrix flowability andmoldability.

A biocompatible matrix comprising a bone scaffolding material and abiocompatible binder, in some embodiments, can also be provided in apredetermined shape including a block, sphere, or cylinder or anydesired shape, for example a shape defined by a mold or a site ofapplication.

A biocompatible matrix comprising a bone scaffolding material and abiocompatible binder, in some embodiments, is bioresorbable. Abiocompatible matrix, in such embodiments, can be resorbed within oneyear of in vivo implantation. In another embodiment, a biocompatiblematrix comprising a bone scaffolding material and a biocompatible bindercan be resorbed within 1, 3, 6, or 9 months of in vivo implantation. Insome embodiments, a biocompatible matrix comprising a scaffoldingmaterial and a biocompatible binder can be resorbed within 1, 3, or sixyears of in vivo implantation. Bioresorbablity will be dependent on: (1)the nature of the matrix material (i.e., its chemical make up, physicalstructure and size); (2) the location within the body in which thematrix is placed; (3) the amount of matrix material that is used; (4)the metabolic state of the patient (diabetic/non-diabetic, osteoporotic,smoker, old age, steroid use, etc.); (5) the extent and/or type ofinjury treated; and (6) the use of other materials in addition to thematrix such as other bone anabolic, catabolic and anti-catabolicfactors.

Biocompatible Matrix Comprising β-TCP and Collagen

In some embodiments, a biocompatible matrix can comprise a β-TCP bonescaffolding material and a biocompatible collagen binder. β-TCP bonescaffolding materials suitable for combination with a collagen binderare consistent with those provided hereinabove.

A collagen binder, in some embodiments, can comprise any type ofcollagen, including Type I, Type II, and Type III collagens. In oneembodiment, a collagen binder comprises a mixture of collagens, such asa mixture of Type I and Type II collagen. In other embodiments, acollagen binder is soluble under physiological conditions. Other typesof collagen present in bone or musculoskeletal tissues may be employed.Recombinant, synthetic and naturally occurring forms of collagen may beused in the present invention.

A biocompatible matrix, according to some embodiments, can comprise aplurality of β-TCP particles adhered to one another with a collagenbinder. In some embodiments, β-TCP particles for combination with acollagen binder have an average diameter ranging from about 1 μm toabout 5 mm. In other embodiments, β-TCP particles have an averagediameter ranging from about 1 mm to about 2 mm, from about 1 mm to about3 mm, from about 100 μm to about 5 mm, from about 100 μm to about 3 mm,from about 250 μm to about 2 mm, from about 250 μm to about 750 μm, fromabout 250 μm to about 1 mm, from about 250 μm to about 2 mm, or fromabout 200 μm to about 3 mm. In another embodiment, β-TCP particles havean average diameter ranging from about 100 μm to about 300 μm. In someembodiments, β-TCP particles have an average diameter ranging from about75 μm to about 300 μm. In some embodiments, β-TCP particles have anaverage diameter of less than about 25 μm, less than about 1 μm, or lessthan about 1 mm. In some embodiments, β-TCP particles have an averagediameter ranging from about 1 nm to about 1 μm. In a further embodiment,β-TCP particles have an average diameter less than about 500 nm or lessthan about 250 nm.

β-TCP particles, in some embodiments, can be adhered to one another bythe collagen binder so as to produce a biocompatible matrix having aporous structure. In some embodiments, the porous structure of abiocompatible matrix comprising β-TCP particles and a collagen binderdemonstrates multidirectional and interconnected pores of varyingdiameters. In some embodiments, a the biocompatible matrix comprises aplurality of pockets and non-interconnected pores of various diametersin addition to the interconnected pores.

In some embodiments, a biocompatible matrix comprising β-TCP particlesand a collagen binder can comprise pores having diameters ranging fromabout 1 μm to about 1 mm. A biocompatible matrix comprising β-TCPparticles and a collagen binder can comprise macropores having diametersranging from about 100 μm to about 1 mm or greater, mesopores havingdiameters ranging from about 10 μm to 100 μm, and micropores havingdiameters less than about 10 μm.

A biocompatible matrix comprising β-TCP particles and a collagen bindercan have a porosity greater than about 25% or greater than about 40%. Inanother embodiment, the biocompatible matrix can have a porosity greaterthan about 50%, greater than about 65%, greater than about 70%, greaterthan about 75%, greater than about 80%, or greater than about 85%. In afurther embodiment, the biocompatible matrix can have a porosity greaterthan about 90%. In some embodiments, the biocompatible matrix can have aporosity that facilitates cell migration into the matrix.

In some embodiments, the β-TCP particles can individually demonstrateany of the pore diameters, pore structures, and porosities providedherein for a biocompatible matrix comprising the β-TCP and collagenbinder.

A biocompatible matrix comprising β-TCP particles, in some embodiments,can comprise a collagen binder in an amount ranging from about 5 weightpercent to about 50 weight percent of the matrix. In other embodiments,a collagen binder can be present in an amount ranging from about 10weight percent to about 40 weight percent of the biocompatible matrix.In another embodiment, a collagen binder can be present in an amountranging from about 15 weight percent to about 35 weight percent of thebiocompatible matrix. In a further embodiment, a collagen binder can bepresent in an amount of about 20 weight percent of the biocompatiblematrix.

A biocompatible matrix comprising β-TCP particles and a collagen binder,according to some embodiments, can be flowable, moldable, and/orextrudable. In such embodiments, the biocompatible matrix can be in theform of a paste or putty. A paste or putty can be molded into thedesired implant shape or can be molded to the contours of theimplantation site. In one embodiment, a biocompatible matrix in paste orputty form comprising β-TCP particles and a collagen binder can beinjected into an implantation site with a syringe or cannula.

In some embodiments, a biocompatible matrix in paste or putty formcomprising β-TCP particles and a collagen binder can retain a flowableand moldable form when implanted. In other embodiments, the paste orputty can harden subsequent to implantation, thereby reducing matrixflowability and moldability.

A biocompatible matrix comprising β-TCP particles and a collagen binder,in some embodiments, can be provided in a predetermined shape such as ablock, sphere, or cylinder.

A biocompatible matrix comprising β-TCP particles and a collagen bindercan be resorbable. In one embodiment, a biocompatible matrix comprisingβ-TCP particles and a collagen binder can be at least 75% resorbed oneyear subsequent to in vivo implantation. In another embodiment, abiocompatible matrix comprising β-TCP particles and a collagen bindercan be greater than 90% resorbed one year subsequent to in vivoimplantation.

A solution comprising PDGF can be disposed in a biocompatible matrix toproduce a composition for treating structures of the vertebral columnaccording to embodiments described herein.

In some embodiments, compositions comprising a PDGF solution disposed ina biocompatible matrix for promoting bone formation in a vertebral bodyand preventing or reducing the likelihood of vertebral compressionfractures, as described herein, further comprise at least one contrastagent. Contrast agents, according to some embodiments, comprise cationiccontrast agents, anionic contrast agents, nonionic contrast agents ormixtures thereof. In some embodiments, contrast agents compriseradiopaque contrast agents. Radiopaque contrast agents, in someembodiments, comprise iodo-compounds including(S)—N,N′-bis[2-hydroxy-1-(hydroxymethyl)-ethyl]-2,4,6-triiodo-5-lactamidoisophthalamide(Iopamidol) and derivatives thereof.

Disposing PDGF Solution in a Biocompatible Matrix

The present invention provides methods for producing compositions forpromoting bone formation in a vertebral body and preventing or reducingthe likelihood of compression fractures of vertebral bodies, includingsecondary vertebral fractures. In one embodiment, a method for producingsuch compositions comprises providing a solution comprising PDGF,providing a biocompatible matrix, and disposing the solution in thebiocompatible matrix. PDGF solutions and biocompatible matrices suitablefor combination are consistent with those described hereinabove.

In some embodiments, a PDGF solution can be disposed in a biocompatiblematrix by soaking the biocompatible matrix in the PDGF solution. A PDGFsolution, in another embodiment, can be disposed in a biocompatiblematrix by injecting the biocompatible matrix with the PDGF solution. Insome embodiments, injecting a PDGF solution can comprise disposing thePDGF solution in a syringe and expelling the PDGF solution into thebiocompatible matrix to saturate the biocompatible matrix.

In some embodiments, the PDGF is absorbed into the pores of thebiocompatible matrix. In some embodiments, the PDGF is adsorbed onto oneor more surfaces of the biocompatible matrix, including surfaces withinpores of the biocompatible matrix.

The biocompatible matrix, according to some embodiments, can be in apredetermined shape, such as a brick or cylinder, prior to receiving aPDGF solution. Subsequent to receiving a PDGF solution, thebiocompatible matrix can have a paste or putty form that is flowable,extrudable, and/or injectable. In other embodiments, the biocompatiblematrix can already demonstrate a flowable paste or putty form prior toreceiving a solution comprising PDGF. Flowable, extrudable, and/orinjectable forms of compositions comprising a PDGF solution disposed ina biocompatible matrix are advantageous for use in methods of thepresent application as they can applied to vertebral bodies withsyringes and/or cannulas.

In some embodiments, methods of producing compositions for promotingbone formation in vertebral bodies and preventing or decreasing thelikelihood of compression fractures in vertebral bodies further compriseproviding at least one contrast agent and disposing the at least onecontrast agent in the biocompatible matrix. In some embodiments,disposing at least one contrast agent in a biocompatible matrixcomprises combining the at least one contrast agent with a PDGF solutionand injecting the biocompatible matrix with the PDGF/contrast agentsolution.

In another embodiment, disposing at least one contrast agent in abiocompatible matrix comprises combining the at least one contrast agentwith a PDGF solution and soaking the biocompatible matrix in thePDGF/contrast agent solution. Alternatively, in some embodiments, acontrast agent is disposed in a biocompatible matrix independent of thePDGF solution.

Contrast agents, according to some embodiments of the present invention,facilitate placement or application of compositions of the presentinvention in and around vertebral bodies. Contrast agents, according tosome embodiments, comprise cationic contrast agents, anionic contrastagents, nonionic contrast agents, or mixtures thereof. In someembodiments, contrast agents comprise radiopaque contrast agents.Radiopaque contrast agents, in some embodiments, comprise iodo-compoundsincluding(S)—N,N′-bis[2-hydroxy-1-(hydroxymethyl)-ethyl]-2,4,6-triiodo-5-lactamidoisophthalamide(Iopamidol) and derivatives thereof.

Compositions Further Comprising Biologically Active Agents

Compositions of the present invention, according to some embodiments,can further comprise one or more biologically active agents in additionto PDGF. Biologically active agents that can be incorporated intocompositions of the present invention, in addition to PDGF, can compriseorganic molecules, inorganic materials, proteins, peptides, nucleicacids (e.g., genes, gene fragments, small interfering ribonucleic acids[si-RNAs], gene regulatory sequences, nuclear transcriptional factors,and antisense molecules), nucleoproteins, polysaccharides (e.g.,heparin), glycoproteins, and lipoproteins. Non-limiting examples ofbiologically active compounds that can be incorporated into compositionsof the present invention, including, e.g., anti-cancer agents,antibiotics, analgesics, anti-inflammatory agents, immunosuppressants,enzyme inhibitors, antihistamines, hormones, muscle relaxants,prostaglandins, trophic factors, osteoinductive proteins, growthfactors, and vaccines, are disclosed in U.S. patent application Ser. No.11/159,533 (Publication No: 20060084602). In some embodiments,biologically active compounds that can be incorporated into compositionsof the present invention include osteostimulatory factors such asinsulin-like growth factors, fibroblast growth factors, or other PDGFs.In accordance with other embodiments, biologically active compounds thatcan be incorporated into compositions of the present inventionpreferably include osteoinductive and osteostimulatory factors such asbone morphogenetic proteins (BMPs), BMP mimetics, calcitonin, orcalcitonin mimetics, statins, statin derivatives, fibroblast growthfactors, insulin-like growth factors, growth differentiating factors,small molecule or antibody blockers of Wnt antagonists (e.g. sclerostin,DKK, soluble Wnt receptors), and/or parathyroid hormone. In someembodiments, factors also include protease inhibitors, as well asosteoporotic treatments that decrease bone resorption includingbisphosphonates, teriparadide, and antibodies to the activator receptorof the NF-kB ligand (RANK) ligand.

Standard protocols and regimens for delivery of additional biologicallyactive agents are known in the art. Additional biologically activeagents can introduced into compositions of the present invention inamounts that allow delivery of an appropriate dosage of the agent to theimplant site. In most cases, dosages are determined using guidelinesknown to practitioners and applicable to the particular agent inquestion. The amount of an additional biologically active agent to beincluded in a composition of the present invention can depend on suchvariables as the type and extent of the condition, the overall healthstatus of the particular patient, the formulation of the biologicallyactive agent, release kinetics, and the bioresorbability of thebiocompatible matrix. Standard clinical trials may be used to optimizethe dose and dosing frequency for any particular additional biologicallyactive agent.

A composition of the present invention, according to some embodiments,can further comprise the addition of additional bone grafting materialswith PDGF including autologous bone marrow, autologous plateletextracts, allografts, synthetic bone matrix materials, xenografts, andderivatives thereof.

Methods of Treating Vertebral Bodies

In some embodiments, the present invention provides methods forpromoting bone formation in a vertebral body comprising providing acomposition comprising a PDGF solution disposed in a biocompatiblematrix and applying the composition to at least one vertebral body. Insome embodiments, the composition can be applied to a plurality ofvertebral bodies. Applying the composition, in some embodiments,comprises injecting at least one vertebral body with the composition.Compositions of the present invention, in some embodiments, are injectedinto the cancellous bone of a vertebral body. Vertebral bodies, in someembodiments, comprise thoracic vertebral bodies, lumbar vertebralbodies, or combinations thereof. Vertebral bodies, in some embodiments,comprise cervical vertebral bodies, coccygeal vertebral bodies, thesacrum, or combinations thereof.

In another aspect, the present invention provides methods for preventingor decreasing the likelihood of vertebral compression fractures,including secondary vertebral compression fractures by strengtheningvertebrae. Preventing or decreasing the likelihood of vertebralcompression fractures, according to embodiments of the presentinvention, comprises providing a composition comprising a PDGF solutiondisposed in a biocompatible matrix and applying the composition to atleast one vertebral body. In some embodiments, applying the compositionto at least one vertebral body comprises injecting the composition intothe at least one vertebral body.

In some embodiments, a composition of the present invention is appliedto a second vertebral body subsequent to vertebroplasty or kyphoplastyof a first vertebral body. In some embodiments, the second vertebralbody is adjacent to the first vertebral body. In other embodiments, thesecond vertebral body is not adjacent to the first vertebral body. In afurther embodiment, a composition of the present invention is applied toa third vertebral body subsequent to vertebroplasty or kyphoplasty of afirst vertebral body. In some embodiments, the third vertebral body isadjacent to the first vertebral body. In other embodiments, the thirdvertebral body is not adjacent to the first vertebral body. Embodimentsof the present invention additionally contemplate application ofcompositions provided herein to a plurality of vertebral bodies,including high risk vertebral bodies, subsequent to vertebroplasty orkyphoplasty of a first vertebral body. It is to be understood thatfirst, second, and third vertebral bodies, as used herein, do not referto any specific position in the vertebral column as methods forinhibiting vertebral compression fractures, including secondarycompression fractures, can be applied to all types of vertebral bodiesincluding thoracic vertebral bodies, lumbar vertebral bodies, cervicalvertebral bodies, coccygeal vertebral bodies, and the sacrum.

In some embodiments, methods for promoting bone formation in vertebralbodies and preventing or decreasing the likelihood of compressionfractures of vertebral bodies further comprise providing at least onepharmaceutical composition in addition to the composition comprising aPDGF solution disposed in a biocompatible matrix and administering theat least one pharmaceutical composition locally and/or systemically. Theat least one pharmaceutical composition, in some embodiments, comprisesvitamins, such as vitamin D3, calcium supplements, or any osteoclastinhibitor known to one of skill in the art, including bisphosphonates.In some embodiments, the at least one pharmaceutical composition isadministered locally. In such embodiments, the at least onepharmaceutical composition can be incorporated into the biocompatiblematrix or otherwise disposed in and around a vertebral body. In otherembodiments, the at least one pharmaceutical composition is administeredsystemically to a patient. In one embodiment, for example, the at leastone pharmaceutical composition is administered orally to a patient. Inanother embodiment, the at least one pharmaceutical composition isadministered intravenously to a patient.

The following examples will serve to further illustrate the presentinvention without, at the same time, however, constituting anylimitation thereof. On the contrary, it is to be clearly understood thatresort may be had to various embodiments, modifications and equivalentsthereof which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the invention.

Example 1 Preparation of a Composition Comprising a Solution of PDGF anda Biocompatible Matrix

A composition comprising a solution of PDGF and a biocompatible matrixwas prepared according to the following procedure.

A pre-weighed block of biocompatible matrix comprising β-TCP andcollagen was obtained. The β-TCP comprised β-TCP particles having anaverage diameter ranging from about 100 μm to about 300 μm. The β-TCPparticles were formulated with about 20% weight percent soluble bovinetype I collagen binder. Such β-TCP/collagen biocompatible matrix can becommercially obtained from Kensey Nash (Exton, Pa.).

A solution comprising rhPDGF-BB was obtained. rhPDGF-BB is commerciallyavailable from Novartis Corporation at a stock concentration of 10 mg/ml(i.e., Lot # QA2217) in a sodium acetate buffer. The rhPDGF-BB isproduced in a yeast expression system by Chiron Corporation and isderived from the same production facility as the rhPDGF-BB that isutilized in the products REGRANEX, (Johnson & Johnson) and GEM 21S(BioMimetic Therapeutics) which have been approved for human use by theUnited States Food and Drug Administration. This rhPDGF-BB is alsoapproved for human use in the European Union and Canada. The rhPDGF-BBsolution was diluted to 0.3 mg/ml in the acetate buffer. The rhPDGF-BBsolution can be diluted to any desired concentration according toembodiments of the present invention, including 1.0 mg/ml.

A ratio of about 3 ml of rhPDGF-BB solution to about 1 g dry weight ofthe β-TCP/collagen biocompatible matrix was used to produce thecomposition. In the preparation of the composition, the rhPDGF-BBsolution was expelled on the biocompatible matrix with a syringe, andthe resulting composition was blended into a paste for placement into asyringe for subsequent injection into a vertebral body.

Example 2 Method of Inhibiting Secondary Vertebral Compression Fractures

Experimental Design and Overview

This prospective, randomized, controlled, single-center clinical trialis to evaluate the efficacy of compositions comprising a PDGF solutiondisposed in a tricalcium phosphate matrix for inhibiting secondarycompression fractures in high risk vertebral bodies (HVBs) at the timeof kyphoplasty of vertebral compression fractures. Comparisons are madebetween vertebral bodies treated with a β-tricalcium phosphate+rhPDGF-BBcomposition and untreated vertebral bodies. The present study is apilot, clinical trial to support the proof-or-principle ofβ-TCP+rh-PDGF-BB to prevent or decrease the likelihood of secondaryvertebral compression fractures by increased bone formation in HVBs.

The study is performed on up to a total of 10 patients requiringprophylactic treatment of HVBs at the time of kyphoplasty. Potentialpatients are screened to determine if they meet the inclusion andexclusion criteria. If all entry criteria are achieved, the potentialpatients are invited to participate in the clinical trial. All patientsconsidered for entry into the study are documented on the Screening Logand reasons for exclusion are recorded.

All patients have undergone kyphoplasty and do not have a symptomaticVCF adjacent to the two vertebral bodies treated in this study. Thesubject is not to be enrolled into the study if the surgeon determinesintraoperatively that the fracture does not meet the fracture enrollmentcriteria or other fractures exist that would preclude treatment in thisprotocol.

A total of 10 patients are enrolled and treated in the present study. Afirst vertebral body adjacent to the kyphoplasty of each patient isinjected with 3.0 ml of a composition of β-TCP+0.3 mg/ml of rhPDGF-BBprepared in accordance with Example 1 herein. The second vertebral bodyadjacent to the kyphoplasty remains untreated and serves as a control.The treated vertebral body may be either cranial or caudal to thekyphoplasty and is determined randomly.

Patients are treated according to the standard protocols and follow-upfor kyphoplasty/vertebroplasty. Each patient is examined by the surgeonat 7-14 days, and at 6, 12, 24, and 52 weeks for clinical, radiographicand quantitative computed tomography (QCT). All over-the-counter andprescribed medication usage is recorded. An independent radiologist,unaware of the patients' treatment group assignments, performs QCTanalysis to assess bone density. These measurements are documented andanalyzed.

All postoperative complications and device-related adverse events arerecorded on the appropriate case report form. If a subject experiences asubsequent VCF during the study period or another surgical procedure fora serious adverse event or the investigational device is removed, thesubject is monitored for safety until the end of the study. Thosesubjects who are re-operated and/or have the fracture fixation hardwareremoved are requested to give permission to examine the explants forhistological purposes. All patients are monitored during the 12-monthtrial and any subject who requests study withdrawal or is withdrawn bythe investigator is requested to provide a reason for studydiscontinuance. Table 1 provides a timeline summary for the presentstudy.

TABLE 1 Study Timeline Survey Visit 1 Visit 2 Screening Surgical VisitVisit ↓ ↓ Visit 3 Visit 4 Visit 5 Visit 6 Visit 7 Within 21 Within 21Post Tx Post Tx Post Tx Post Tx Post Tx Days of Days of Follow Up FollowUp Follow Up Follow Up Follow Up Surgery Screening ↓ ↓ ↓ ↓ ↓ Day 0 Day7-14 Week 6 ± Week 12 ± Week 24 ± Week 52 ± 3 days 7 days 7 days 14 days

The primary endpoint is the bone density at 12 weeks post-operativelymeasured by QCT scans. Secondary endpoints include subject pain andquality of life assessments.

Surgical Protocol

After patients have been enrolled in the study, satisfying both theinclusion and exclusion criteria, the following surgical protocol isundertaken.

Patients are brought into an operating room (OR) in the standardfashion, and standard methods are used to perform the kyphoplastyprocedure with methyl methacrylate cement augmentation of the fracturedvertebral body. Standard radiographs are taken of the vertebral bodiestreated with kyphoplasty and with preventative bone augmentationtreatment.

Following the kyphoplasty treatment, the investigator identifies andqualifies the two levels to be treated with prophylactic boneaugmentation. If two (2) qualified vertebral bodies are not availablefor treatment, as determined at the time of surgery, the patient isconsidered a screen failure and not enrolled into the study.

Upon identification of the two HVBs, the investigator requests that therandomization code be opened to determine the study treatmentadministered. The randomization code specifies treatment with theβ-TCP+rhPDGF composition either proximally or distally in relation tothe level treated with kyphoplasty. The other HVB remains untreated.

The β-TCP+rhPDGF composition is mixed according to the procedureprovided in Example 1. Once mixed, the paste is loaded into a syringefor injection using aseptic technique. Once the β-TCP+rhPDGF compositionis mixed, the clinician waits about 10 minutes prior to implantation. Anew sterile mixing device (spatula) is used for each mix. Theinvestigator directs the assistant who performs the mixing to record thecumulative amount of implanted composition, as well as the residualamount of composition not implanted. The amount of composition iscalculated and documented using qualitative relative measurements (⅓, ⅔,All).

An 8 to 16 gauge JAMSHIDI® needle available from Cardinal Health ofDublin, Ohio is inserted through an extrapedicular approach into thevertebral bodies requiring prophylactic treatment. The wire is passedthrough the JAMSHIDI® needle and the JAMSHIDI® needle through the styletover the wire. The appropriate mixed preparation is injected into thesubject vertebral body. Care should be taken to minimize leakage of thepaste outside of the vertebral body.

Contrast agents, according to embodiments of the present invention, canassist in identifying the leakage of the paste outside the vertebralbody. FIG. 1 illustrates a syringe and related apparatus penetratingtissue overlaying a vertebral body to deliver a composition of thepresent invention to the vertebral body. FIG. 2 is a radiographillustrating injection of a composition of the present invention intothe vertebral body of the L3 vertebra according to one embodiment.

The instrumentation is removed. Thorough irrigation and standard woundclosure techniques are employed.

Follow-Up Evaluations

Patients are seen for post-operative evaluations at days 7-14, and at 6(±3 days), 12 (±7 days), 24 (±7 days), and 52 (±14 days) weekspost-surgery. Routine evaluations and procedures are performed duringthe follow-up period, as specified in the study flowchart of Table 2below.

TABLE 2 Study Flow Chart and Follow-up Assessments Post-TreatmentFollow-Up Evaluations Surgery Visit Visit Visit Visit Screening VisitVisit 4 5 6 7 Visit 2 3 Week 6 ± Week 12 ± Week 24 ± Week 52 ± Procedure1 Day 0 Day 7-14 3 Days 7 Days 7 Days 14 Days Informed Consent  X¹Screening Log X Medical History X Physical Examination of X X X X X X XSpine Subject Eligibility X X Criteria Verification Identification ofHigh- X Risk Vertebral Bodies Randomization X Kyphoplasty and XPreventative Bone Augmentation Volume of Graft Material X PlacedQualitative CT X X X X Assessments² Plain Radiographic X X X X X XAssessments Adverse X X X X X X Events/Complications ConcomitantMedications X X X X X X X Review ¹Must occur prior to any study-specificprocedures. ²Quantitative Computed Tomography (QCT) is performedaccording to standard protocol to obtain BMD data which is determined bythe designated musculoskeletal radiologist.Assessment of Effectiveness

Outcome data is collected from this study on findings derived fromradiographs, QCTs, and from direct examination of function. The scheduleof these measurements is provided in Table 3.

TABLE 3 Frequency of Radiographic and Functional Assessments StudyParameters Plain film Qualitative CT Timepoint radiographs Scans PainFunction Prior to X X X X Treatment Immediately X Post-Treatment Day7-14 X Week 6 X X X X Week 12 X X X X Week 24 X X X X Week 52 X X X X

Vertebral bodies injected with a β-TCP+rhPDGF composition are expectedto display increased bone mineral density (BMD) in comparison tountreated vertebral bodies. Increased bone mineral density in avertebral body can render the vertebral body less susceptible tofractures including secondary fractures induced bykyphoplasty/vertebroplasty operations.

Example 3 Method of Inhibiting Vertebral Compression Fractures inOsteoporotic Individuals

A method of inhibiting vertebral compression fractures in osteoporoticindividuals comprises promoting bone formation in vertebral bodiesthrough treatment with compositions comprising a PDGF solution disposedin a biocompatible matrix such as β-tricalcium phosphate.

Compositions of the present invention are mixed in accordance with thatprovided in Example 1. The concentration of PDGF in the PDGF solutionsranges from 0.3 mg/ml to 1.0 mg/ml. Once mixed, the composition isloaded into a syringe for injection using aseptic technique. The surgeonwaits about 10 minutes prior to implantation. A new sterile mixingdevice (spatula) is used for each mix.

The JAMSHIDI® needle is inserted through an extrapedicular approach intothe vertebral bodies requiring prophylactic treatment. Vertebral bodiesrequiring prophylactic treatment, in some embodiments, comprise highrisk vertebral bodies including vertebral bodies T5 through T12 and L1through L4. The wire is passed through the JAMSHIDI® needle and theJAMSHIDI® needle through the stylet over the wire. The mixed compositionis injected into the subject vertebral body. Care is taken to minimizeleakage of the paste outside of the vertebral body. A plurality ofvertebral bodies are treated according to the present example.Osteoporotic patients receiving this treatment have a lower incidence ofvertebral compression fractures than untreated osteoporotic patients.

Example 4 Evaluation of the Chronic Safety of Rh-PDGF-BB Combined withCollagen/β-Tricalcium Phosphate Matrix in a Rabbit Paravertebral ImplantModel

Experimental Design and Overview

This study evaluated the safety of implanting injectablerhPDGF-BB/collagen/β-TCP material in a paravertebral intramuscular siteadjacent to the spine of rabbits. The animals were observed for signs ofneurotoxicity, and the implant sites with adjacent vertebral bodies andspinal cord were examined histologically to document tissue-specificresponses to the material.

The study protocol and animal care was approved by the local IACUC andconducted according to AAALAC guidelines. Twelve (12) naïve, female,albino New Zealand rabbits weighing ≥2.5 kg were assigned to one of 4groups: 0.3 mg/ml PDGF; 1.0 mg/ml PDGF; rubber; or acetate buffer. PDGFtreated rabbits received 0.2 cc implants of appropriately concentratedrhPDGF-BB in matrix injected into a 1 cm pocket in the rightparavertebral muscle adjacent to the L4-L5 vertebral bodies while highdensity polyethylene (HDPE) was implanted in a similar incision in theleft paravertebral muscles near L2-L3 of the same animals. Rabbits inthe sodium acetate buffer group received sodium acetate buffer in placeof the PDGF+matrix implant, while those in the rubber group receivedonly rubber in the right paravertebral muscle. One rabbit in each groupwas sacrificed at 29, 90, and 180 days post-surgery.

Body weights were measured prior to surgery and biweekly followingsurgery for the duration of the study. Radiographs were taken prior tosurgery, immediately following surgery, and immediately prior tosacrifice. Digital photography of the surgical sites was performedduring surgery and at the study end points. Weekly clinical observationsof the implant sites were recorded for signs of erythema, edema, andinflammation and for signs of neurotoxicity, such as ambulatory changes.At necropsy, each implant site along with the adjacent vertebral bodyand spinal cord were harvested en bloc, fixed in formalin, and preparedfor decalcified, paraffin embedded histopathological analysis.

Materials

The dosages of rhPDGF-BB tested in this study included 0.3 mg/ml and 1.0mg/ml in 20 mM sodium acetate buffer, pH 6.0+/−0.5. The matrix materialconsisted of 20% lyophilized bovine type I collagen and 80% β-TCP with aparticle size of 100-300 μm (Kensey Nash Corporation). Negative controlmaterial consisted of high-density polyethylene (HDPE) and positivecontrol material consisted of black rubber. Immediately prior tosurgery, the rhPDGF-BB and control solutions were mixed with matrixmaterial in a 3:1 liquid to mass ratio.

Briefly, the PDGF solution was allowed to saturate the material forabout 2 minutes then was manually mixed for about 3 minutes to generatea paste-like consistency. The homogeneous distribution of rhPDGF-BBthroughout the mixed material using this mixing technique was confirmedby eluting the PDGF from samples of similar mass and then quantifyingthe PDGF by ELISA (R&D Systems).

Results

Following manual mixing of 0.3 mg/ml rhPDGF-BB with the collagen/β-TCPmatrix, the homogeneity of rhPDGF-BB throughout the mixed material wasconfirmed within +/−4% error across samples.

All animals recovered from surgery, and at the time of this writing, allclinical observations were reported to be normal with no signs ofneurotoxicity or abnormal wound healing at the surgical sites. Twoanimals treated with sodium acetate buffer and matrix control exhibitedminor scabbing at the surgical wounds which healed completely. Oneanimal that received 0.3 mg/ml rhPDGF-BB exhibited slight erythema atthe surgical site 3-4 days after surgery and then returned to normalappearance. A histopathological analysis of test article implant sites29 days post-surgery indicated a mild amount of tissue in-growth intothe implanted test materials and a mild inflammatory response. Noectopic or abnormal bone formation was observed in the vertebral bodiesadjacent to the implant sites. These findings are summarized in Table 4and compared with ratings for negative control HDPE implant sites.

TABLE 4 Summary of Histopathology Findings at Implant Sites 29 DaysAfter Surgery [PDGF-BB] Macro- Tissue In- Ectopic (mg/ml) phages MGCsgrowth Bone Exostosis 0.3 3, 1(NC) 2, 0(NC) 2, 0(NC) 0, 0(NC) 0, 0(NC)1.0 2, 2(NC) 2, 0(NC) 2, 0(NC) 0, 0(NC) 0, 0(NC) NC = Negative Control;MGC = multinucleated giant cells; Bioreactivity scale: 0 = Absent, 1 =Minimal/Slight, 2 = Mild, 3 = Moderate, 4 = Marked/Severe

Preliminary evidence from this study based on clinical observations,suggests that collagen/β-tricalcium phosphate combined with either 1.0mg/ml, 0.3 mg/ml rhPDGF-BB, or sodium acetate buffer does not elicit anyacute or chronic neurotoxic effects. Histopathological assessment of theimplant sites 29 days post-surgery indicated a normal and expected mildamount of tissue in-growth into the implanted material and a mildinflammatory response. No ectopic bone formation, exostosis, or abnormalbone resorption was observed at any of the implant sites. Based onobservations of the animals treated in this study, collagen/β-tricalciumphosphate combined with either 1.0 mg/ml, 0.3 mg/ml rhPDGF-BB is safe touse when injected in close proximity to the spinal column.

Example 5 Evaluation of the Safety of PDGF-BB Combined with a BovineType I Collagen/β-TCP Matrix for Vertebral Therapy

This study evaluated the safety of a composition comprising rhPDGF-BBcombined with a biocompatible matrix comprising β-tricalcium phosphateand type I collagen for bone augmentation following injection of thecomposition into vertebral bodies of baboons. Experimental Design

A total of 6 female baboons (Papio anubis) of 18 to 21 years of age werestudied, each baboon being assigned to one of two treatment groups asprovided in Table 5. During the study, the animals were imaged andanalyzed using radiography, quantitative computed tomography (QCT),magnetic resonance imaging (MRI) techniques, terminal histology andnon-GLP microcomputed tomography (microCT).

Four vertebral levels (T12, L2, L4 and L6) were investigated in eachanimal. Each animal of Group I received an injection of about 0.5 cc ofa 1.0 mg/ml rhPDGF-BB+collagen/β-TCP (matrix) composition into each ofthe T12, L2 and L4 vertebral bodies. The 1.0 mg/mlrhPDGF-BB+collagen/β-TCP (matrix) compositions were prepared as setforth in Example 1 hereinabove. Each animal of Group II received aninjection of about 0.5 cc of a sodium acetate buffer+collagen/β-TCP(matrix) composition into each of the T12, L2, and L4 vertebral bodies.Animals of each group I and II additionally received an injection ofabout 0.5 cc of a sodium acetate buffer into the L6 vertebral bodies.Therefore, a total of four (4) vertebral bodies per animal received aninjection. FIG. 3 summarizes the injection strategy of the presentstudy. Documentation of the treatments in each animal was recorded onstudy forms.

Surgery was conducted using a percutaneous, fluoroscopically guidedapproach. The procedure was performed similar to a vertebroplasty,except that an injectable 1.0 mg/ml rhPDGF-BB+collagen/β-TCP matrix orappropriate control treatment was injected. About 0.5 cc of arhPDGF-BB+collagen/β-TCP material, control material, or buffer wasinjected into each vertebral body as described above. Each animal wasprovided anesthesia during the surgery.

TABLE 5 Summary of Treatments for Injection of rhPDGF-BB +Collagen/β-TCP Matrix in Vertebral Bodies of Baboons Group DoseTimepoints Analyses I 1.0 mg/ml PDGF + Pre- and post- QCT, MRI, ClinicalObservations, collagen/β-TCP surgery, 1 and 3 Serum Chemistry andHematology matrix. L6 received months, (optionally 6 (Pre- andpost-surgery, and at 1 sodium acetate buffer months) month, 3 months,and 6 months only post-surgery), Body Weights, Radiography, Histology,non- GLP microCT II 20 mM sodium Pre- and post- QCT, MRI, ClinicalObservations, acetate buffer, pH 6.0 + surgery, 1 and 3 Serum Chemistryand Hematology collagen/β-TCP months, (optionally 6 (Pre- andpost-surgery, and at 1 matrix. L6 received months) month, 3 months, and6 months sodium acetate buffer post-surgery), Body Weights, onlyRadiography, Histology, non- GLP microCTA. Assignment to Dose Group

Three animals were assigned to treatment groups by a manual schemedesigned to achieve similar group mean body weights.

B. Assignment to Surgery Days

Animals were assigned to one of two surgery days (Day I or Day II). Foreach animal, a coin flip determined assignment into the Day I group orthe Day II group. This process was continued until each of the days wasfilled with three animals. The animal numbers, their dosing groupassignments, and surgery days were recorded. The animal treatment groupswere known to the study monitors and study director. The radiologistsand histopathologist were blind to the treatment groups.

C. In-Vivo Observations and Measurements

Clinical Observations

Animals were observed within their cages daily throughout the study.Recording of cageside observations was commenced after the pre-selectioncriteria was completed and is continued until the end of study. Eachanimal was observed for changes in general appearance and behavior,including changes in ambulation. Each animal was observed for evidenceof menstrual cycling over the course of the study. The cycling readingswere performed non-GLP and were recorded in the Southwest Foundation forBiomedical Research (SFBR) animal database.

Treatment of the animals was in accordance with SFBR standard operatingprocedures (SOPs), which adhere to the regulations outlined in the USDAAnimal Welfare Act (9 CFR, Parts 1, 2 and 3) and the conditionsspecified in The Guide for Care and Use of Laboratory Animals (ILARpublication, 1996, National Academy Press). The study animals wereobserved and were recorded at least once daily for signs of illness ordistress, including changes in ambulation, and any such observationswere reported to the responsible veterinarian and study director.

Body Weight

Body weights were measured at the initial health check, prior tosurgery, and prior to follow up radiographs. Food was withheld prior tosedation and subsequent body weight measurements.

Food Consumption

Except when animals were fasting for study procedures, food consumptionwas qualitatively assessed daily for each animal (as part of thecageside observations), beginning at least 7 days prior to surgery. Eachanimal was provided a full feeder of food once a day (non-sedation days)and the amount eaten was documented as per SFBR SOPs. On sedation days,the animals were fed once each day when they recovered from anesthesia.

D. Fluoroscopy, Photography, Radiography, MRI and QCT Imaging

Non-GLP digital photographs were taken of the injection sitespre-operatively, immediately post-operatively, and at 1, 3, 6, and 9months post-operatively.

Anteroposterior and lateral radiographs were taken pretreatment andimmediately following surgery as well as at about 1, 3, 6, and 9 monthspost-operatively. For anteroposterior radiographs, the animals wereplaced on their backs in supine position with their legs supported. Forlateral radiographs, the animals were positioned lying on their leftsides with arms and legs supported. Energy (kV) and intensity (mA)settings for each position and animal were recorded.

Non-GLP fluorographs were captured intraoperatively before, during, andafter injection of the test and control articles into the vertebrae ofthe animals. Fluorographs were not assessed as an outcome of this study,but enable the surgeon to accurately insert the introduction needle intothe vertebral bodies during surgery.

Magnetic resonance imaging (MRI) was performed to image the spine ofeach animal pre-operatively and within 4 to 10 days post-operatively.MRI was additionally performed to image the spine of each animal atabout 1, 3, 6, and 9 months post-operatively. MRI sessions consisted ofT1 and T2-weighted scans.

Quantitative computed tomography imaging (QCT) was performed to imagethe spine of each animal pre-operatively and within 4 to 10 dayspost-operatively. QCT was additionally performed to image the spine ofeach animal at about 1, 3, 6, and 9 months post-operatively. Scansconsisted of a series of contiguous cross-sectional slices of the torsofrom the caudal endplate of the 11th thoracic vertebrae to the cranialendplate of the sacrum.

QCT was also used to obtain 3 mm thick cross sectional images ofinjected and intervening untreated vertebral bodies of each baboon 1week prior to surgery (pre-surgical) and at 1, 4, and 12 weeks postsurgery. A total of 5-8 slices were required to fully image eachvertebral body. The DICOM (digital imaging and communications inmedicine) format images produced by the QCT were transferred andconverted to a file format for 3-dimensional volumetric analysis usingsoftware developed by Scanco AG (Bassersdorf, Switzerland). Volumetricbone mineral density (vBMD) of the anterior compartment of eachvertebral body was determined by manually selecting a region of interest(ROI) that excluded the cortical shell in each slice. The evaluationsoftware created a z-stack of the individual slices and ROI's beforeextracting the volumetric ROI and calculating volumetric density inarbitrary units derived from the gray-scale intensity in the images anda percent change from the baseline scans was calculated. One-wayrepeated measures ANOVA with Tukey's post-hoc test was used to determinethe presence of any statistically significant changes in vBMD foranimals of Group I and Group II from pre-surgical or 1 wk post-surgicalto the conclusion of the study.

The radiographs, MRI, and QCT images were evaluated by one boardcertified clinical radiologist and one qualified associate to provide aconsensus assessment of the neuropathological, osteopathological andsurrounding soft tissue pathological outcomes resulting from thetreatments of the vertebrae. The evaluation consisted of a qualitativeexamination of each image for abnormalities of the bone and of theneural tissues and adjacent surrounding soft tissues. The radiologistfollowed the Radiology Assessment Protocol to evaluate the radiologydata.

E. Clinical Pathology Evaluation

Serum Chemistry

About 3 ml whole blood was collected into containers withoutanticoagulant pre-surgery and post-surgery. About 3 ml whole blood wasalso collected into containers without anticoagulant at about 1, 3, 6,and 9 months post-operatively. The animals were fasted overnight priorto blood collection for serum chemistry. Serum was analyzed for thefollowing parameters set forth in Table 6.

TABLE 6 Serum Analysis Sodium Phosphorus Potassium Glucose Chloride Ureanitrogen (BUN) Total carbon dioxide (bicarbonate) Creatinine Totalbilirubin Total protein Alkaline phosphatase (AP) Albumin Lactatedehydrogenase (LDH) Globulin Aspartate aminotransferase (AST)Albumin/globulin ratio Alanine aminotransferase (ALT) CholesterolGamma-glutamyltransferase (GGT) Triglycerides Calcium BUNICREAT RatioAnion Gap Direct Bilirubin CPKHematology

About 3 ml of blood was collected in EDTA-containing tubes pre-surgeryand post-surgery. About 3 ml of blood was also collected inEDTA-containing tubes at 1, 3, 6, and 9 months post-surgery. The wholeblood samples were analyzed for the following parameters set forth inTable 7.

TABLE 7 Blood Analysis Red blood cell (RBC) counts Mean corpuscularhemoglobin (MCH) White blood cells (WBCs) Mean corpuscular hemoglobin(total and differential*) concentration (MCHC) Hemoglobin concentrationMean corpuscular volume (MCV) Hematocrit Platelet counts (Plt) RDWAbnormal blood cell morphology *Includes polysegmented neutrophils, bandcells, lymphocytes, monocytes, basophils, eosinophils.Serum Collection for Analysis by Sponsor or SFBR

About 14 ml of blood was collected into non-additive (i.e., “clot”)tubes from all animals once pre-surgery and post-surgery. About 14 ml ofblood was also collected into non-additive (i.e., “clot”) tubes from allanimals at 1, 3, 6, and 9 months post-surgery. The blood was centrifugedto obtain serum and divided into two aliquots. The serum is stored at−70° C. or lower.

Anatomic Pathology

All animals are humanely sacrificed at the end of the study. A grossnecropsy is conducted on each animal sacrificed in a moribund ordiseased condition to determine the cause and/or nature of the moribundor diseased condition.

Necropsy

A complete necropsy is conducted under the supervision of the studypathologist on the sacrificed animals in a moribund or diseasedcondition during the study to determine the cause and/or nature of themoribund or diseased condition. A standard necropsy includes anexamination of external surfaces and orifices, extremities, bodycavities, and internal organ/tissues. All of the treated vertebrae arecollected and are examined for abnormalities. A brief morphologicdescription of all macroscopic abnormalities is recorded on individualnecropsy forms.

Tissue Collection and Preservation

The following tissues and organs are obtained at sacrifice and arepreserved in 10% neutral-buffered formalin (except for the eyes, whichwere preserved in Bouin's Solution for optimum fixation). Each tissue ororgan specimen is then embedded in paraffin for preservation purposesand is archived at a sponsor-approved site or used to help determine thecause of death.

For all baboons, all treated and adjacent untreated vertebrae (T12 thruL6) are individually harvested en bloc including the spinal cord andspinal canal and are appropriately identified as to the treatmentreceived. The T12 vertebral body is identified by leaving a minimum of 2cm of the ribs attached to the bone. All bone specimens are placed informalin fixative in preparation for plastic embedding.

Each of the soft tissues or organ specimens is embedded in paraffin forpreservation purposes and are archived. A summary of the tissue samplesto be collected is provided in Table 8.

TABLE 8 Summary of Tissue Samples to be Collected at NecropsyCardiovascular Urogenital Aorta Kidneys Heart Urinary Bladder DigestiveOvaries Salivary Gland (mandibular) Uterus Tongue Cervix EsophagusVagina Stomach Skin/Musculoskeletal Small Intestine Skin/Mammary Gland(males and females) Duodenum Bone (femoral head) Jejunum Bone (7th rib)Ileum Skeletal Muscle (thigh) Large Intestine Knee joint Cecum Shoulderjoint Colon Mandible Rectum Right Foot Pancreas Left Ankle Liver RightHand Gallbladder Left Wrist Respiratory Spine from T12 to L7/Sacrum -separated Trachea Nervous/Special Sense Lung (including bronchi) Eyeswith optic nerve Lymphoid/Hematopoietic Sciatic Nerve Bone Marrow(sternum) Brain Thymus Optic chiasm Spleen Cerebrum Lymph NodesCerebellum Axillary Medulla Mandibular Pons Mesenteric Spinal Cord(thoracic) Endocrine Other Adrenals Animal Number Tattoo Pituitary GrossLesions Thyroid/Parathyroids* Lacrimal glands *The occasional absence ofthe parathyroid gland from the routine tissue section will not require arecut of the section.

Vertebral bodies injected with a composition comprising a rhPDGF-BBsolution disposed in β-TCP/collagen matrix displayed the formation ofnormal bone with no adverse neurotoxic effects. Moreover, soft tissuesadjacent to vertebral bodies receiving a composition comprising arhPDGF-BB solution disposed in β-TCP/collagen matrix did not demonstrateabnormalities resulting from the administration of the rh-PDGF/matrixcomposition.

Furthermore, vertebral bodies injected with a composition comprising arhPDGF-BB solution disposed in β-TCP/collagen matrix additionallydisplayed increased volumetric bone mineral densities. FIG. 4illustrates percent change in volumetric bone mineral density (vBMD) forvertebral bodies of animals of Group I and Group II. Each data point inFIG. 4 represents an average of all vertebral bodies treated within eachgroup. For example, the first data point in FIG. 4 for Group I is theaverage of nine vertebral body measurements (T12, L2, and L4 for each ofthree animals in Group I) taken after injection of the rhPDGF-BB matrixcomposition into the vertebral bodies. Similarly, the first data pointin FIG. 4 for Group 2 is the average of nine vertebral body measurements(T12, L2 and L4 for each of three animals in Group II) taken afterinjection of a collagen/β-TCP matrix into the vertebral bodies.

As illustrated in FIG. 4, vertebral bodies treated with a compositioncomprising a rhPDGF-BB solution disposed in β-TCP/collagen matrix (GroupI) demonstrated a steady increase in vBMD over the course of the study,the increase in vBMD becoming statistically significant over the 1 weekpost-operative level by the third month [2.64%+/−1.16 (week 1) v.5.93%+/−1.33 (week 12); p=0.023]. vBMD continued to increase through thesixth month of the study before reaching a plateau at the ninth month.Vertebral bodies treated with a composition comprising 20 mM sodiumacetate buffer disposed in the β-TCP/collagen matrix (Group II),however, did not demonstrate significant increases in vBMD over thecourse of the study.

Additionally, FIG. 5 illustrates percent change in vBMD for vertebralbodies of animals of Group I and Group II wherein the injectedβ-TCP/collagen matrix is subtracted from the volumetric bone mineraldensity analysis. As in FIG. 4, each data point in FIG. 5 represents anaverage of all vertebral bodies treated within each group.

As illustrated in FIG. 5, vertebral bodies treated with a rhPDGF-BBmatrix composition (Group I) demonstrated increases in vBMD. Thesubtraction of the β-TCP/collagen matrix from the volumetric bonemineral density analysis provided a clear indication that vBMD increasedthroughout all regions of the vertebral bodies of Group I as opposed toregions local to the injection site of the rhPDGF-BB matrix composition.

All patents, publications and abstracts cited above are incorporatedherein by reference in their entirety. It should be understood that theforegoing relates only to preferred embodiments of the present inventionand that numerous modifications or alterations may be made thereinwithout departing from the spirit and the scope of the present inventionas defined in the following claims.

The invention claimed is:
 1. A method for promoting or accelerating boneformation in at least one vertebral body of a patient comprising:applying to the vertebral body a composition comprising: a biocompatiblematrix having incorporated therein a solution of platelet derived growthfactor (PDGF) at a concentration in a range of about 0.1 mg/ml to about1.0 mg/ml in a buffer, wherein the biocompatible matrix comprises (i)particles of a porous calcium phosphate in a range of about 100 μm toabout 3 mm in size or (ii) (a) particles of a porous calcium phosphatein a range of about 100 μm to about 3 mm in size and (b) collagen,wherein the calcium phosphate comprises interconnected pores and aporosity greater than 50%.
 2. The method of claim 1, wherein thebiocompatible matrix comprises the particles of porous calcium phosphateand collagen.
 3. The method of claim 2, wherein the collagen comprisesType I collagen.
 4. The method of claim 2, wherein the weight ratio ofcalcium phosphate:collagen is about 80:20.
 5. The method of claim 1,wherein the PDGF is at a concentration of about 0.3 mg/ml.
 6. The methodof claim 1, wherein the PDGF comprises PDGF-BB.
 7. The method of claim6, wherein the PDGF comprises recombinant human PDGF-BB (rhPDGF-BB) or afragment thereof.
 8. The method of claim 7, wherein the rhPDGF-BBcomprises at least 65% of intact rhPDGF-BB.
 9. The method of claim 7,wherein the fragment of rhPDGF-BB is selected from the group consistingof amino acid sequences 1-31, 1-32, 33-108, 33-109 and 1-108 of theentire B chain.
 10. The method of claim 1, wherein the calcium phosphatecomprises particles in a range of about 100 μm to about 300 μm in size.11. The method of claim 1, wherein the calcium phosphate comprisesparticles in a range of about 1000 μm to about 2000 μm in size.
 12. Themethod of claim 1, wherein the calcium phosphate comprises particles ina range of about 250 μm to about 1000 μm in size.
 13. The method ofclaim 1, where the calcium phosphate comprises β-tricalcium phosphate.14. The method of claim 2, wherein the composition is flowable.
 15. Themethod of claim 14, wherein applying the composition to the vertebralbody comprises injecting the composition into the vertebral body. 16.The method of claim 1, wherein the method prevents or inhibits vertebralcompression fractures.
 17. The method of claim 1, further comprisinglocally or systemically administering to the patient at least oneadditional pharmaceutical composition.
 18. The method of claim 17,wherein the at least one additional pharmaceutical composition comprisesa vitamin or an osteoclast inhibitor.
 19. The method of claim 17,wherein the locally administering comprises disposing the at least oneadditional pharmaceutical composition in or around the at least onevertebral body.
 20. The method of claim 19, wherein the patient hasundergone kyphoplasty or vertebroplasty.
 21. The method of claim 17,wherein the systemically administering comprises orally administering,intravenously administering, or a combination thereof.